Cell death in an organism is under genetic control. Genetically controlled cell death, also known as programmed cell death, has been best described during fetal development. As a normal part of development of an adult organism, cells that are not needed die via activation of a cascade of death-promoting genes. Meanwhile, other genes are expressed during development that promote survival of cells that are required in the adult. Besides elimination of unwanted cells during development, many adult cells turn over. For example, gastroepithelial cells die and are replaced by new cells constantly. This physiologic cell death also occurs via the process of programmed cell death. Many cells that undergo programmed cell death develop stereotypic morphologic changes referred to as apoptosis.
Yet other genes that control cell death have been discovered by cancer researchers. Cancer is a state where there are defects in genes that control normal programmed cell death. Mutations in a series of cancer causing genes (oncogenes) are necessary for the development of cancer. Thus, many genes that control programmed cell death are also oncogenes.
In the last several years it has become apparent that the genes that regulate programmed cell death are also important in cell death under pathologic conditions. Thompson, Science 267:1456 (1995). For example, in brain, expression of genes that regulate cell death is increased after such pathologic insults as ischemia and epilepsy. Chen, J., et al., J. Clin. Pathol. 48:7 (1995). Furthermore, DNA laddering, a biochemical hallmark of programmed cell death, and some of the morphologic changes that characterize apoptosis occur in these disease states. Linnik, M.D., et al., Stroke 24:2002 (1993). Alteration of expression of such death regulatory genes by various means alters outcome in cerebral ischemia. For example, the death regulatory gene bcl-2 is an oncogene which suppresses cell death. Its expression is induced after cerebral ischemia. Chen, J., et al., Neuroreport 6:394 (1993). Transgenic mice which express the human bcl-2 transgene have been shown to have smaller strokes after middle cerebral artery occlusion than wild-type mice. Martinou, J. C. et al., Neuron 13:1017 (1994). Furthermore, over expression of bcl-2 prevents programmed cell death of neurons in culture. Kane, et al., Science 262:1274 (1993).
Other genes that promote programmed cell death have also been implicated in ischemia and other neurologic diseases. For example, the cysteine proteases are a family of genes that promote programmed cell death during development. Cysteine protease inhibitors decrease the volume of infarction after middle cerebral occlusion in rats. Hara, et al., PNAS USA 94:2007 (1997). Thus, there is evidence that the expression and activity of these death regulatory genes may contribute to stroke and other neurologic diseases.
Transcription factors are proteins that bind to nuclear double stranded DNA and regulate the expression of other genes. Homeotic proteins (homeobox or HOX proteins) are a family of transcription factors that share a highly conserved amino acid sequence (the homeodomain) that is the site where the protein binds to DNA. The homeodomain is conserved from primitive organisms such as the nematode C. elegans to humans. The amino acid sequences that flank the highly conserved amino acid sequence are not conserved. These flanking regions determine which genes the respective homeobox proteins regulate. There have been over forty different human homeobox proteins identified. All of these proteins bind to a specific DNA sequence, the homeodomain-binding site. HOX genes regulate cell and organ differentiation during development. Wilson, et al., Curr. Biol. 5:32 (1995).
Programmed cell death requires a cascade of gene expression events. Early steps in this cascade include the expression of transcription factors. Englekamp, et al., Curr. Opin. Genet. Dev. 6:334 (1996). There are two examples of homeobox genes that regulate cell death as reported by Williams, G. J., et al., Cell 74:777 (1993). The apoptotic cell death of C. elegans somatic neurons is induced by the homeobox gene Lin-34. Clark, S. G., et al., Cell 74:43 (1993). Additionally, expression of some human homeobox genes is altered in neoplastic cells. Nakamura, et al., Nat. Genet. 12:154 (1996).
The mechanism by which neurons die in many disease states is not well understood. Furthermore, means of diagnosis of these conditions and methods of treatment are not available. Therefore, there remains a need for further elucidation of the genes that promote or suppress cell death, particularly in brain. Cloning and sequencing of genes whose expression is induced in brain cells, specifically hippocampal neurons, after global ischemia are needed to permit development of techniques by which such genes, their expression products, and antibodies therefor can be employed in studies, diagnoses, and therapies for treatment of disorders such as stroke, epilepsy, neurodegenerative diseases, and cancer.